Nearly 30 million patients in North America and Europe have been diagnosed with peripheral artery disease (PAD). While all patients suffer from quality-of-life issues due to decreased limb perfusion, unmanaged PAD ultimately leads to about 250,000 annual low extremity amputations in the western world. Surgical and endovascular revascularization is performed in an effort to reopen occluded vessels, but 40% of patients with critical limb ischemia (CLI) do not qualify for these procedures due to extreme tissue damage and/or diffuse atherosclerotic disease. Even with revascularization, the rate of amputation and five-year mortality rate remain high. Thus, these patients have a critically unmet need for limb salvage therapy. Biomaterials have shown promise in the treatment of numerous diseases, including PAD. We were the first to show a decellularized skeletal muscle ECM hydrogel improved perfusion, arteriogenesis, and muscle regeneration in rat hindlimb ischemia injury. In side-by-side comparisons of skeletal muscle ECM and a human umbilical cord ECM, only the skeletal muscle ECM improved skeletal muscle morphology and regeneration, suggesting a tissue-specific effect in muscle-dependent regeneration. Others have shown that tissue specific decellularized materials impact tissue specific cell types in vitro, but has not been definitively studied in vivo. Thus, the goal of this proposal is to mechanistically interrogate tissue specificity of ECM hydrogels on skeletal muscle regeneration through quantitative systems-level analysis. We will be the first group to demonstrate quantitatively and functionally the impact of properly controlled tissue specific ECM hydrogels in a complex in vivo microenvironment. We hypothesize that the tissue specific source of decellularized extracellular matrix will differentially impact whole muscle function, genetic pathway activation, and the spatiotemporal muscle regenerative response in vivo. We propose to develop improved ECM hydrogels for PAD to revascularize and regenerate damaged skeletal muscle with the following specific aims: Aim 1: Demonstrate impact of decellularized hydrogel tissue specificity on functional whole muscle contractility and blood perfusion. Aim 2: Determine effects of tissue specific ECM hydrogels on transcriptomic and spatiotemporal cellular responses in muscle regeneration. This project will use a myriad of multi-scale tools, such as biomaterial fabrication techniques, in vivo animal studies, muscle functional testing, bioinformatics, and immunohistochemistry to accomplish the aims and develop the PI for a career in scientific research. With an aging population the incidence of PAD is expected to increase with no synchronous improvements in treatment strategies. We have the potential to reduce this extreme outcome through mechanistic understanding of minimally invasive, injectable, and easily fabricated decellularized materials.